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ANIMAL GROWTH, PHYSIOLOGY, AND REPRODUCTION |
Department of Obstetrics & Gynaecology, Faculty of Medicine, National University of Singapore, Singapore 119074
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Abstract |
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Key Words: Development • In Vitro Maturation • Meiosis • Porcine • Temperature
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
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; Coy et al., 1999). However, one major drawback is the possible exposure of the GV stage oocytes to elevated temperatures during the slaughter procedure. In some slaughterhouses, dehairing is achieved by hot water treatment followed by flame sterilization before the carcass is cut open to remove the entrails. Therefore, this study investigated whether such exposure to elevated temperature could compromise the maturational and developmental competence of such GV-stage oocytes, as a preliminary assessment of suitability for embryo production. |
Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
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, and the experimental protocol was approved by the Ethics Committee for Experimental Animals of the National University of Singapore.
Media and Reagents
Unless otherwise stated, all reagents and chemicals used in this study were obtained from Sigma Inc. (St Louis, MO). Human recombinant (Gonal-F) and hCG (Profasi) were obtained from Serono Inc. (Aubonne, Switzerland). Tissue culture medium 199 (M199) was obtained from Gibco BRL, Inc. (Auckland, New Zealand). The NCSU23 medium supplemented with 0.4% (wt/vol) BSA was prepared according to the method of Petters and Wells (1993). The four-well dishes used in all experiments were purchased from Nunc, Inc. (Copenhagen, Denmark).
Processing of Adult Sows during Slaughter
Adult sows (Landrace x Large White x Duroc, 6 to 8 mo of age, 75 to 115 kg BW) were slaughtered by electrical shock and dehaired by vertical hot water shower at 65°C for 10 min, followed by flame sterilization. Hair removal by vertical hot water and flame sterilization was not applied to controls. A microcomputer thermometer probe (Hanna Instruments, Woonsocket, RI) was inserted through the rectum, into the pelvic cavity near the ovary to measure the pelvic temperature during postslaughter processing. Temperature readings were taken at two time-points: immediately after postslaughter processing and 30 min later. This resulted in a mean value of 42.1 ± 0.9°C (n = 35 sows) immediately after postslaughter processing, and a mean value of 41.3 ± 0.8°C (n = 35 sows) when temperature readings were taken 30 min later. For the nonheat-treated controls, the pelvic temperature was within the physiological range, with a mean value of 39.4 ± 0.5°C (n = 40 sows).
Oocyte Collection and In Vitro Maturation
Ovaries were collected from the abattoir and washed in PBS supplemented with 50 µg/L of penicillin and 75 µg/L of streptomycin. The ovaries were maintained at 30 to 35°C in PBS during transport to the laboratory. Cumulus–oocyte complexes (COC) were aspirated from antral follicles (3 to 7 mm diameter) using a beveled 16-gauge needle fixed to a 10-mL syringe. Only COC with at least three layers of granulosa cells were selected. These were washed four times in M199 supplemented with 20 mM HEPES, 1g/L polyvinyalcohol, 50 µg/L of penicillin, and 75 µg/L of streptomycin, to remove all debris and blood.
Oocyte maturation in vitro was carried out in four-well Nunclon dishes containing 0.5 mL of equilibrated culture medium overlaid with mineral oil (embryo tested). The culture medium used was M199 supplemented with 10% (vol/vol) follicular fluid, 1 mM glutamine, 0.03 mM sodium pyruvate, 0.1 IU/mL of FSH, 0.5 IU/mL of hCG, 0.57 µM cysteine, 50 µg/L of penicillin, and 75 µg/L of streptomycin. After 36 h of in vitro culture in a 5% CO2 incubator set at 39°C, the COC were denuded by repeated pipetting with 80 IU/mL of hyaluronidase in 20 mM HEPES-buffered M199. A total of 263 and 215 GV stage oocytes were allocated to the heat-treated and nonheat-treated control groups, respectively.
Assessment of Nuclear Maturation
Completely denuded oocytes were stained with 10 µg/mL of bisbenzimide (Hoechst 33342) for 5 min and viewed under UV light with a Hoffman-modulation contrast microscope at 100x magnification. Two distinct spots of fluorescence would be observed for mature metaphase II (MII) stage oocytes. These corresponded to the nucleus of the first polar body and the chromosomes of the MII spindle of the mature oocyte.
Visualization of Oocyte Metaphase II Spindle, Chromosomal DNA, and Microfilament by Immunocytochemical Staining and Confocal Laser Microscopy
Mature MII-stage oocytes were subjected to immunocytochemical staining before being visualized under confocal laser microscopy, according to the technique described by Kim et al. (1996). Oocytes with an obvious first polar body were permeabilized in Buffer M (25% glycerol, 50 mM KCl, 0.5 mM MgCl2, 0.1 mM EDTA, 1 mM 2-ß mercaptoethanol, 50 mM imdazole, 3% triton X-100, and 25 mM phenylmethylsulfoxy fluoride) for 20 min at 25°C, fixed in methanol at –20°C, and then kept in storage medium (0.02% sodium azide, 0.01% BSA and PBS) for up to 7 d at 4°C. For immunocytochemical staining, the oocytes were incubated with the first antibody (mouse monoclonal antialpha-tubulin antibody, 1:300 dilution) for 1 h at 39°C, before being washed in PBS and incubated in blocking medium (0.1 M glycine, 1% goat serum, 0.01% Triton X-100, 1% skim milk, 0.05% BSA, 0.02% sodium azide, and PBS) for 1 h at 39°C. Subsequently, the oocytes were incubated with the second antibody (fluorescein isothiocyanate-coated goat anti-mouse antibody, 1:200 dilution) at 39°C for 1 h and then washed in PBS. The 
-tubulin of the MII spindle that was bound to the fluorescein isothiocyanate-conjugated antibody would appear green under a laser wavelength of 488 nm. After rinsing, the oocytes were double-stained with either 50 µg/mL of propidium iodide (45-min incubation at 39°C) to detect chromosomal DNA (Figure 1), or with 15 IU/mL of phalloidin-tetramethylrhodamine (1 h incubation at 39°C) to detect microfilament (Figure 2). Both these would fluoresce red, with an excitation wavelength of 260 nm for propidium iodide, and an excitation wavelength of 488 nm for phalloidin-tetramethylrhodamine. For double staining to detect microtubulin and chromosomal DNA, a total of 30 and 35 MII-stage oocytes were allocated to the heat-treated and nonheat-treated control groups, respectively. For double staining to detect microtubulin and microfilament, a total of 15 and 18 MII-stage oocytes were allocated to the heat-treated and nonheat-treated control groups, respectively. Oocytes that were subjected to double staining did not represent a subset of the larger numbers depicted in Table 1.
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Statistical Analyses
The COC collected from 10 to 20 ovaries were randomly allocated to the different treatment groups within each set of experiments. Ten replicates were carried out for each treatment group, with each replicate representing a different trip to the abattoir. The nuclear maturation rate was expressed as a percentage of the initial number of GV-stage oocytes cultured within each treatment group, whereas the cleavage and blastulation rates were expressed as a percentage of the number of MII-stage oocytes subjected to parthenogenetic activation. The 
2 test was used to compare nuclear maturation, cleavage, and blastulation rates in the different treatment groups. A probability of P < 0.05 was considered statistically significant.
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Results |
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). Only 27.7% of mature oocytes from heat-treated sows cleaved after parthenogenetic activation, of which 6.7% developed to the blastocyst stage on d 6. For oocytes obtained from nonheat-treated control sows, the corresponding cleavage and blastulation rates were significantly higher, at 65.3 and 20.6%, respectively (P < 0.01). Hence, exposure to elevated temperature during postslaughter processing did not adversely affect oocyte nuclear maturation per se, but severely compromised the subsequent developmental competence.
The 30 MII-stage oocytes from heat-treated sows and 35 MII-stage oocytes from nonheat-treated sows were assessed for metaphase II spindle structure and distribution of cytoskeletal elements (microtubules/micro-filaments). Determination of a normal MII spindle was based on the criteria established by Kim et al. (1996). As seen in Figure 1, a normal MII spindle would have a distinct barrel shape, with the chromosomal DNA (red fluorescence) concentrated as a distinct band within the center, and the -tubulin (green fluorescence) localized at the periphery. Twenty-nine of 30 mature oocytes from heat-treated sows and all 35 mature oocytes from nonheat-treated controls showed normal metaphase II spindle formation, indicating that nuclear maturation had proceeded normally (Figure 1). The spindles and chromosomes of both groups of oocytes appeared similar, with barrel-shaped spindle and chromosomes aligned on the metaphase plate. Oocytes with atypical spindle or misaligned chromosomes were considered abnormal, and this was seen in only one heat-treated oocyte. Therefore, the results confirm that exposure to elevated temperature did not adversely affect MII spindle formation.
Further immunocytochemical staining revealed that transient exposure to elevated temperature resulted in severe disruption of oocyte cytoskeletal elements. The microtubule and microfilament elements of all 15 non-heat-treated control oocytes were concentrated in the cortex and uniformly distributed in the ooplasm. In contrast, the microtubule and microfilament elements of all 18 heat-treated oocytes seemed to be decreased or even lost in the ooplasm (Figure 2).
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Discussion |
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) and cattle (Ju et al., 1999; Tseng et al., 2004) revealed that a slightly elevated temperature of 41.5°C had detrimental effects on the structural integrity of nuclear and cytoskeletal elements within the oocyte, as well as on its subsequent developmental competence. Nevertheless, these studies were based solely on mature MII oocytes exposed to heat shock in vitro. To complement their data, our study attempted to study the effects of exposing immature GV oocytes to elevated temperatures in vivo.
In this study, oocytes from heat-treated and nonheat-treated control sows presented similar IVM rates (approximately 70 to 75%) under identical in vitro culture condition. These values were comparable to those obtained in earlier studies (Wu et al., 2001; Abeydeera, 2001, 2002; Kikuchi et al., 2002). Additionally, immunocytochemical staining showed that exposure to elevated temperature did not adversely affect the formation of the MII spindle. Hence, it is obvious that exposure to elevated temperatures during postslaughter processing did not compromise nuclear maturation per se.
In contrast, the parthenogenetic developmental competence of heat-treated oocytes was significantly lower than that of the nonheat-treated controls. This is probably due to disruption of oocyte cytoskeletal elements on exposure to elevated temperature, as evidenced by immunocytochemical staining (Figure 2). In this study, parthenogenetic activation was used to assess oocyte developmental competence, instead of in vitro fertilization. This is because zona hardening during oocyte in vitro maturation (Zhang et al., 1991; Dell’Aquila et al., 2001; Coy et al., 1999, 2002) could lead to an artifactual decrease in fertilization rates. Additionally, it has also been reported that boar spermatozoa do not survive well during cryopreservation (Thurston et al., 2001, 2002). In vitro fertilization is therefore not a reliable means of assessing the developmental competence of porcine IVM oocytes.
Hence, it seems that meiotic progression is not as sensitive to elevated temperatures as developmental competence. The process of oocyte maturation comprises both a nuclear and cytoplasmic component. It can therefore be surmised that nuclear maturation was not affected; rather, it was cytoplasmic maturity that was compromised by exposure to elevated temperature, as evidenced by the disrupted cytoskeletal organization of heat-treated oocytes. The integral role of cytoskeletal organization in embryonic development is also borne out by the study of Phillips et al. (2004); they demonstrated that a dominant missense mutation of -tubulin gene tba-1 in Caenorhabditis elegans led to a disruption of pronuclear migration and positioning of the first mitotic spindle. This in turn resulted in high embryonic mortality. It seems that once the meiotic process is set in motion, it may continue in the face of minor disruptions such as transient exposure to elevated temperature. The spindle structure formed during the metaphase I and II stages is made up of centrosomal matrix proteins that are relatively heat-stable (De Carcer et al., 2001) and microtubules that are stabilized by phosphorylation. (Mandelkow and Mandelkow, 1995; Liang and MacRae, 1997; Mayor et al., 1999). Presumably, these components are not disrupted by exposure to a mild elevation in temperature, so that the process of nuclear maturation from the GV to MII stage was unaffected. Our data (Table 1) indicated that it was the first cleavage division to the two-cell stage that was dramatically compromised, rather than later embryonic development to the blastocyst stage. When the blastulation rates were computed as a percentage of two-cell stage embryos, no significant difference between the embryos from the heat-treated sows vs. the corresponding nonheat-treated controls (24.2 vs. 31.5%) was observed. This clearly supports the idea that it was cytoskeletal damage sustained during exposure to elevated temperature that was primarily responsible for the decrease in oocyte developmental competence. One possible conjecture is that if the heat-treated oocyte was able to cleave, this could either mean that intrinsic mechanisms within the oocyte had repaired the cytoskeletal damage, or that the sustained damage was less severe compared with other heat-treated oocytes that failed to cleave.
In conclusion, transient exposure to elevated temperatures during postslaughter processing of sows drastically reduced the quality of recovered GV-stage oocytes. Such oocytes are therefore unsuitable for large-scale embryo production.
1 Correspondence: Embryonics Int., 6A Napter Rd., No. 01-38, Singapore 258500 (phone: +65-6479-7267; fax: +65-6479-6536; e-mail: scng{at}embryonics.biz).
Received for publication May 22, 2004. Accepted for publication July 14, 2004.
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